Three-dimensional (3D) scaffolds are the choice materials for mimicking the natural environment of many cell types, which in vivo are normally integrated into well-structured and dense structures. Thus, 3D materials for cell culture and for studies on cell adhesion and cell migration have become highly relevant in many applications.
The architectural features in terms of pore morphology (pore size, shape, pore surface characteristics, porosity and interconnectivity) are manifestly important for the performance of such 3D environments. A wide range of techniques have been proposed to design 3D matrices, e.g. selective laser sintering, multiphoton lithography/direct laser writing, stereolithography, bioplotting or 3D printing/fused deposition modeling. In general these techniques are quite complex, expensive and do not offer great flexibility in terms of materials to be used and/or of obtainable structures.
A further common technique to generate 3D environments consists of reverse fabrication or pore-leaching, where pores are introduced into a bulk material by dissolving salt crystals or other dissolvable particles that were previously embedded in that bulk material. Such techniques have the disadvantage that the pores are mainly inverse-opal shaped, so that the interconnectivity of the pores is dependent on the pore size and density, i.e. a high degree of interconnectivity implies large pore sizes. For many applications, however, it is advantageous to maximize the contact between the cells and the scaffold by using pores that are approximately equal to the cell size. Through this, a large contact area of cell and the pore surface is ensured. Under such circumstances, the impact of material parameters such as stiffness or functionalization is much higher than in materials with large pores. U.S. Pat. No. 6,673,285 teaches the reverse fabrication of porous materials with a pre-designed three-dimensional negative replica of the desired pore configuration. The methods disclosed therein are reported to generate materials with high porosity and interconnected large pores, generally larger than cell size. However, this patent does not teach specifically how pore interconnectivity may be achieved independently from pore density and how to define shape and size of the matrix, its porosity as well as its stiffness independently from each other.
There is an ongoing need for 3D structured materials that mimic complex 3D structures found in vivo as well as for efficient, easy and inexpensive methods for fabricating such 3D materials. In particular there is a need for methods to generate scaffolds with flexible, easily customizable porosity characteristics and with pores that are approximately equal to the cell size.
The use of porous scaffolds as cell traps has been proposed as a means to eliminate undesirable cells in vivo. For example, a method to recruit and eliminate metastatic cancer cells, wherein cancer cells migrate and accumulate in a porous matrix is described in WO 2014063128. Another example is the device comprising a porous scaffold composition which attracts, adheres, captures and eliminates targeted undesirable cells as disclosed in U.S. patent application Ser. No. 12/665,761. In both cases, bioactive agents in the scaffold compositions are used to attract and/or destroy the undesirable cells.
Acanthamoeba castellanii (A. castellanii) are free-living protists often found in tap water and swimming pools. If transmitted to the eye they can cause acanthamoeba keratitis, which has become a serious disease among contact lens users. An estimated 85% of acanthamoeba keratitis cases are related to contact lens usage (Patel et al. Current Opinion in Ophthalmology 2008, 19, 302-306). Until 2003, more than 2000 cases of this extremely painful partial destruction of the cornea have been reported (Walochnik et al. Wien Klin Wochenschr. 2003, 115, 10), and even further cases occurred during the 2004 to 2007 outbreak of acanthamoeba keratitis in multiple states of the US (Johnston et al. Journal of Clinical Microbiology 2009, 47, 2040-2045). Infections of contact lens users with A. castellanii are mainly due to wrong contact lens care, but have also been associated with the resistance of A. castellanii cysts to contact lens cleaning solutions.
Using a combined treatment of a special multipurpose solution and peroxide treatment minimizes the risk for A. castellanii growth even on lens materials with high water content, yet not all hydrogen peroxide solutions on the market kill A. castellanii. Although some studies also suggest that silver nanoparticles are a promising strategy to kill A. castellanii and thus prevent infection, recent studies have shown that even for small silver concentrations, cytotoxicity against mammalian cells is present. Thus, it is questionable if silver-coated contact lens storage cases are the right strategy to prevent A. castellanii infections, as this would mean a constant exposure of the eye's epithelial cells to silver ions.
Contact lenses with high water content are currently highly appreciated among contact lens users, but unfortunately A. castellanii adhesion increases with increasing water content of the lens. A way to prevent A. castellanii infections might be to adapt the mechanical properties of the contact lens material. Recent studies have shown that substrate stiffness strongly controls the adhesion and differentiation of mammalian cell types. However, it was shown that this strategy is not suitable for contact lenses, as the mechanical stiffness threshold for A. castellanii adhesion is far below any stiffness value suitable for contact lens materials (Gutekunst et al. Beilstein Journal of Nanotechnology 2014, 5, 1393-1398).
Accordingly, there exists an ongoing need to develop new methods for minimizing the presence of A. castellanii in contact lens environments, thus helping to prevent A. castellanii infections in contact lens users. Sequestering the cells from the contact lens solution, especially in a non-toxic way, e.g. without the use of bioactive agents such as chemoattractants, would be an ideal way to minimize cell proliferation and reduce A. castellanii infections.